Sex differences and immune correlates of Long COVID development, persistence, and resolution.

Sex differences have been observed in acute COVID-19 and Long COVID (LC) outcomes, with greater disease severity and mortality during acute infection in males and a greater proportion of females developing LC. We hypothesized that sex-specific immune dysregulation contributes to the pathogenesis of LC. To investigate the immunologic underpinnings of LC development and persistence, we used single-cell transcriptomics, single-cell proteomics, and plasma proteomics on blood samples obtained during acute SARS-CoV-2 infection and at 3 and 12 months post-infection in a cohort of 45 patients who either developed LC or recovered. Several sex-specific immune pathways were associated with LC. Specifically, males who would develop LC at 3 months had widespread increases in TGF-ß signaling during acute infection in proliferating NK cells. Females who would develop LC demonstrated increased expression of XIST, an RNA gene implicated in autoimmunity, and increased IL1 signaling in monocytes at 12 months post infection. Several immune features of LC were also conserved across sexes. Both males and females with LC had reduced co-stimulatory signaling from monocytes and broad upregulation of NF-κB transcription factors. In both sexes, those with persistent LC demonstrated increased LAG3, a marker of T cell exhaustion, reduced ETS1 transcription factor expression across lymphocyte subsets, and elevated intracellular IL-4 levels in T cell subsets, suggesting that ETS1 alterations may drive an aberrantly elevated Th2-like response in LC. Altogether, this study describes multiple innate and adaptive immune correlates of LC, some of which differ by sex, and offers insights toward the pursuit of tailored therapeutics.

The impact of alcohol and illicit substance use on the pharmacokinetics of first-line TB drugs.

BACKGROUND: In South Africa, an estimated 11% of the population have high alcohol use, a major risk factor for TB. Alcohol and other substance use are also associated with poor treatment response, with a potential mechanism being altered TB drug pharmacokinetics. OBJECTIVES: To investigate the impact of alcohol and illicit substance use on the pharmacokinetics of first-line TB drugs in participants with pulmonary TB. METHODS: We prospectively enrolled participants ≥15 years old, without HIV, and initiating drug-susceptible TB treatment in Worcester, South Africa. Alcohol use was measured via self-report and blood biomarkers. Other illicit substances were captured through a urine drug test. Plasma samples were drawn 1 month into treatment pre-dose, and 1.5, 3, 5 and 8 h post-dose. Non-linear mixed-effects modelling was used to describe the pharmacokinetics of rifampicin, isoniazid, pyrazinamide and ethambutol. Alcohol and drug use were tested as covariates. RESULTS: The study included 104 participants, of whom 70% were male, with a median age of 37 years (IQR 27-48). Alcohol use was high, with 42% and 28% of participants having moderate and high alcohol use, respectively. Rifampicin and isoniazid had slightly lower pharmacokinetics compared with previous reports, whereas pyrazinamide and ethambutol were consistent. No significant alcohol use effect was detected, other than 13% higher ethambutol clearance in participants with high alcohol use. Methaqualone use reduced rifampicin bioavailability by 19%. CONCLUSION: No clinically relevant effect of alcohol use was observed on the pharmacokinetics of first-line TB drugs, suggesting that poor treatment outcome is unlikely due to pharmacokinetic alterations. That methaqualone reduced rifampicin means dose adjustment may be beneficial.

Nirmatrelvir-Ritonavir and Symptoms in Adults With Postacute Sequelae of SARS-CoV-2 Infection: The STOP-PASC Randomized Clinical Trial.

Importance: There is an urgent need to identify treatments for postacute sequelae of SARS-CoV-2 infection (PASC). Objective: To assess the efficacy of a 15-day course of nirmatrelvir-ritonavir in reducing the severity of select PASC symptoms. Design, Setting, and Participants: This was a 15-week blinded, placebo-controlled, randomized clinical trial conducted from November 2022 to September 2023 at Stanford University (California). The participants were adults with moderate to severe PASC symptoms of 3 months or longer duration. Interventions: Participants were randomized 2:1 to treatment with oral nirmatrelvir-ritonavir (NMV/r, 300 mg and 100 mg) or with placebo-ritonavir (PBO/r) twice daily for 15 days. Main Outcomes and Measures: Primary outcome was a pooled severity of 6 PASC symptoms (fatigue, brain fog, shortness of breath, body aches, gastrointestinal symptoms, and cardiovascular symptoms) based on a Likert scale score at 10 weeks. Secondary outcomes included symptom severity at different time points, symptom burden and relief, patient global measures, Patient-Reported Outcomes Measurement Information System (PROMIS) measures, orthostatic vital signs, and sit-to-stand test change from baseline. Results: Of the 155 participants (median [IQR] age, 43 [34-54] years; 92 [59%] females), 102 were randomized to the NMV/r group and 53 to the PBO/r group. Nearly all participants (n = 153) had received the primary series for COVID-19 vaccination. Mean (SD) time between index SARS-CoV-2 infection and randomization was 17.5 (9.1) months. There was no statistically significant difference in the model-derived severity outcome pooled across the 6 core symptoms at 10 weeks between the NMV/r and PBO/r groups. No statistically significant between-group differences were found at 10 weeks in the Patient Global Impression of Severity or Patient Global Impression of Change scores, summative symptom scores, and change from baseline to 10 weeks in PROMIS fatigue, dyspnea, cognitive function, and physical function measures. Adverse event rates were similar in NMV/r and PBO/r groups and mostly of low grade. Conclusions and Relevance: The results of this randomized clinical trial showed that a 15-day course of NMV/r in a population of patients with PASC was generally safe but did not demonstrate a significant benefit for improving select PASC symptoms in a mostly vaccinated cohort with protracted symptom duration. Further studies are needed to determine the role of antivirals in the treatment of PASC. Trial Registration: ClinicalTrials.gov Identifier: NCT05576662.

For Tuberculosis, Not "To Screen or Not to Screen?" but "Who?" and "How?"

Active case finding leveraging new molecular diagnostics and chest X-rays with automated interpretation algorithms is increasingly being developed for high-risk populations to drive down tuberculosis incidence. We consider why such an approach did not deliver a decline in tuberculosis prevalence in Brazilian prison populations and what to consider next.

Analysis of the limited M. tuberculosis accessory genome reveals potential pitfalls of pan-genome analysis approaches.

Pan-genome analysis is a fundamental tool for studying bacterial genome evolution; however, the variety of methods used to define and measure the pan-genome poses challenges to the interpretation and reliability of results. To quantify sources of bias and error related to common pan-genome analysis approaches, we evaluated different approaches applied to curated collection of 151 Mycobacterium tuberculosis ( Mtb ) isolates. Mtb is characterized by its clonal evolution, absence of horizontal gene transfer, and limited accessory genome, making it an ideal test case for this study. Using a state-of-the-art graph-genome approach, we found that a majority of the structural variation observed in Mtb originates from rearrangement, deletion, and duplication of redundant nucleotide sequences. In contrast, we found that pan-genome analyses that focus on comparison of coding sequences (at the amino acid level) can yield surprisingly variable results, driven by differences in assembly quality and the softwares used. Upon closer inspection, we found that coding sequence annotation discrepancies were a major contributor to inflated Mtb accessory genome estimates. To address this, we developed panqc, a software that detects annotation discrepancies and collapses nucleotide redundancy in pan-genome estimates. When applied to Mtb and E. coli pan-genomes, panqc exposed distinct biases influenced by the genomic diversity of the population studied. Our findings underscore the need for careful methodological selection and quality control to accurately map the evolutionary dynamics of a bacterial species.

Monthly Sulfadoxine-Pyrimethamine During Pregnancy Prevents Febrile Respiratory Illnesses: A Secondary Analysis of a Malaria Chemoprevention Trial in Uganda.

Background: Trials evaluating antimalarials for intermittent preventive treatment in pregnancy (IPTp) have shown that dihydroartemisinin-piperaquine (DP) is a more efficacious antimalarial than sulfadoxine-pyrimethamine (SP); however, SP is associated with higher birthweight, suggesting that SP demonstrates "nonmalarial" effects. Chemoprevention of nonmalarial febrile illnesses (NMFIs) was explored as a possible mechanism. Methods: In this secondary analysis, we leveraged data from 654 pregnant Ugandan women without HIV infection who participated in a randomized controlled trial comparing monthly IPTp-SP with IPTp-DP. Women were enrolled between 12 and 20 gestational weeks and followed through delivery. NMFIs were measured by active and passive surveillance and defined by the absence of malaria parasitemia. We quantified associations among IPTp regimens, incident NMFIs, antibiotic prescriptions, and birthweight. Results: Mean "birthweight for gestational age" Z scores were 0.189 points (95% CI, .045-.333) higher in women randomized to IPTp-SP vs IPTp-DP. Women randomized to IPTp-SP had fewer incident NMFIs (incidence rate ratio, 0.74; 95% CI, .58-.95), mainly respiratory NMFIs (incidence rate ratio, 0.69; 95% CI, .48-1.00), vs IPTp-DP. Counterintuitively, respiratory NMFI incidence was positively correlated with birthweight in multigravidae. In total 75% of respiratory NMFIs were treated with antibiotics. Although overall antibiotic prescriptions were similar between arms, for each antibiotic prescribed, "birthweight for gestational age" Z scores increased by 0.038 points (95% CI, .001-.074). Conclusions: Monthly IPTp-SP was associated with reduced respiratory NMFI incidence, revealing a potential nonmalarial mechanism of SP and supporting current World Health Organization recommendations for IPTp-SP, even in areas with high-grade SP resistance. While maternal respiratory NMFIs are known risk factors of lower birthweight, most women in our study were presumptively treated with antibiotics, masking the potential benefit of SP on birthweight mediated through preventing respiratory NMFIs.

Tuberculosis testing patterns in South Africa to identify groups that would benefit from increased investigation.

The National Health Laboratory Service (NHLS) collects all public health laboratory test results in South Africa, providing a cohort from which to identify groups, by age, sex, HIV, and viral suppression status, that would benefit from increased tuberculosis (TB) testing. Using NHLS data (2012-2016), we assessed levels and trends over time in TB diagnostic tests performed (count and per capita) and TB test positivity. Estimates were stratified by HIV status, viral suppression, age, sex, and province. We used logistic regression to estimate the odds of testing positive for TB by viral suppression status. Nineteen million TB diagnostic tests were conducted during period 2012-2016. Testing per capita was lower among PLHIV with viral suppression than those with unsuppressed HIV (0.08 vs 0.32) but lowest among people without HIV (0.03). Test positivity was highest among young adults (aged 15-35 years), males of all age groups, and people with unsuppressed HIV. Test positivity was higher for males without laboratory evidence of HIV than those with HIV viral suppression, despite similar individual odds of TB. Our results are an important national baseline characterizing who received TB testing in South Africa. People without evidence of HIV, young adults, and males would benefit from increased TB screening given their lower testing rates and higher test positivity. These high-test positivity groups can be used to guide future expansions of TB screening.

Integrating Molecular Diagnostics and GIS Mapping: A Multidisciplinary Approach to Understanding Tuberculosis Disease Dynamics in South Africa Using Xpert MTB/RIF.

An investigation was carried out to examine the use of national Xpert MTB/RIF data (2013-2017) and GIS technology for MTB/RIF surveillance in South Africa. The aim was to exhibit the potential of using molecular diagnostics for TB surveillance across the country. The variables analysed include Mycobacterium tuberculosis (Mtb) positivity, the mycobacterial proportion of rifampicin-resistant Mtb (RIF), and probe frequency. The summary statistics of these variables were generated and aggregated at the facility and municipal level. The spatial distribution patterns of the indicators across municipalities were determined using the Moran's I and Getis Ord (Gi) statistics. A case-control study was conducted to investigate factors associated with a high mycobacterial load. Logistic regression was used to analyse this study's results. There was striking spatial heterogeneity in the distribution of Mtb and RIF across South Africa. The median patient age, urban setting classification, and number of health care workers were found to be associated with the mycobacterial load. This study illustrates the potential of using data generated from molecular diagnostics in combination with GIS technology for Mtb surveillance in South Africa. Spatially targeted interventions can be implemented in areas where high-burden Mtb persists.

Patient and provider perceptions of the relationship between alcohol use and TB and readiness for treatment: a qualitative study in South Africa.

Background: Unhealthy alcohol use is widespread in South Africa and has been linked to tuberculosis (TB) disease and poor treatment outcomes. This study used qualitative methods to explore the relationship between TB and alcohol use during TB treatment. Methods: Focus groups (FGs) were conducted with 34 participants who had previous or current drugsusceptible TB and self-reported current alcohol use. Eight interviews were conducted with healthcare workers who provide TB services in Worcester, South Africa. Results: In this rural setting, heavy episodic drinking is normalized and perceived to be related to TB transmission and decreased adherence to TB medication. Both healthcare workers and FG participants recommended the introduction of universal screening, brief interventions, and referral to specialized care for unhealthy alcohol use. However, participants also discussed barriers to the provision of these services, such as limited awareness of the link between alcohol and TB. Healthcare workers also specified resource constraints while FG participants or patients mentioned widespread stigma towards people with alcohol concerns. Both FG participants and health providers would benefit from education on the relationship between TB and unhealthy alcohol use as well and had specific recommendations about interventions for alcohol use reduction. Healthcare workers also suggested that community health worker-delivered interventions could support access to and engagement in both TB and alcohol-related services. Conclusion: Findings support strengthening accessible, specialized services for the identification and provision of interventions and psychosocial services for unhealthy alcohol use among those with TB.

High resolution imaging and five-year tuberculosis contact outcomes.

Background: The evolution of tuberculosis (TB) disease during the clinical latency period remains incompletely understood. Methods: 250 HIV-uninfected, adult household contacts of rifampicin-resistant TB with a negative symptom screen underwent baseline 18F-Fluorodeoxyglucose positron emission and computed tomography (PET/CT), repeated in 112 after 5-15 months. Following South African and WHO guidelines, participants did not receive preventive therapy. All participants had intensive baseline screening with spontaneous, followed by induced, sputum sampling and were then observed for an average of 4.7 years for culture-positive disease. Baseline PET/CT abnormalities were evaluated in relation to culture-positive disease. Results: At baseline, 59 (23.6%) participants had lung PET/CT findings consistent with TB of which 29 (11.6%) were defined as Subclinical TB, and 30 (12%) Subclinical TB-inactive. A further 83 (33.2%) had other lung parenchymal abnormalities and 108 (43.2%) had normal lungs. Over 1107-person years of follow-up 14 cases of culture-positive TB were diagnosed. Six cases were detected by intensive baseline screening, all would have been missed by the South African symptom-based screening strategy and only one detected by a WHO-recommended chest X-Ray screening strategy. Those with baseline Subclinical TB lesions on PET/CT were significantly more likely to be diagnosed with culture-positive TB over the study period, compared to those with normal lung parenchyma (10/29 [34.5%] vs 2/108 [1.9%], Hazard Ratio 22.37 [4.89-102.47, p<0.001]). Conclusions: These findings challenge the latent/active TB paradigm demonstrating that subclinical disease exists up to 4 years prior to microbiological detection and/or symptom onset. There are important implications for screening and management of TB.

Safety and tolerability of isoniazid preventive therapy for tuberculosis for persons with HIV with and without alcohol use.

OBJECTIVE: Isoniazid (INH) preventive therapy is recommended to prevent tuberculosis (TB) disease for persons with HIV (PWH), except for those with regular and heavy alcohol consumption, due to hepatotoxicity concerns. We aimed to quantify the incidence of severe INH-related toxicity among PWH with and without recent alcohol consumption. DESIGN: A prospective study of PWH receiving INH. METHODS: We included PWH in southwest Uganda with recent (prior 3 months) ( n  = 200) or no (prior year) self-reported alcohol consumption ( n  = 101), on antiretroviral therapy, TB infected (≥5 mm on tuberculin skin test), and alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 2× or less the upper limit of normal (ULN). Grade 3+ INH-related toxicity was ALT or AST at least 5× the ULN or severe symptoms; we stopped IPT upon detection. Grade 2 INH-related toxicity was ALT or AST 2-5× the ULN or moderate symptoms. RESULTS: The cumulative incidence of Grade 3+ INH-related toxicity was 8.3% [95% confidence interval (95% CI) 5.4-12.0]; all resolved after INH cessation. Incidence was 6.0% (95% CI 3.1-10.2) among those reporting recent alcohol use and 12.9% (95% CI 7.0-21.0) among those reporting no prior year alcohol use. We found no differences by baseline phosphatidylethanol-confirmed alcohol severity. The cumulative incidence of Grade 2 toxicities (without Grade 3+) was 21.7% (95% CI 17.0-27.1); 25.0% (95% CI 19.0-31.8) among those with recent alcohol use and 14.8% (95% CI 8.1-23.9) among those with no prior year alcohol use. CONCLUSION: Alcohol use does not appear to increase risk for serious INH-related toxicity among PWH without significant liver enzyme elevations at baseline (≤2x ULN).

At Long Last: Short, All-Oral Regimens for Multidrug-Resistant Tuberculosis in the United States.

Multidrug-resistant tuberculosis (MDR-TB) has historically required longer treatment regimens that were associated with higher unfavorable outcomes and side effects rates compared with drug susceptible TB (DS-TB). During the last decade, several studies conducted mostly in high-incidence settings have shown that MDR-TB can be successfully treated using all-oral shorter regimens of 6- to 9-month duration. In this article, we review the evolution of MDR-TB treatment from the early long regimens with injectables agents (IAs), followed by the shorter regimens with IA, to the groundbreaking, all-oral, 6- to 9-month regimens. Finally, we propose a framework for implementation of the shorter all-oral regimens in the United States.

Mycobacterium tuberculosis disease associates with higher HIV-1-specific antibody responses.

Mycobacterium tuberculosis (Mtb) is the most common infection among people with HIV (PWH). Mtb disease-associated inflammation could affect HIV-directed immune responses in PWH. We show that HIV antibodies are broader and more potent in PWH in the presence as compared to the absence of Mtb disease. With co-existing Mtb disease, the virus in PWH also encounters unique antibody selection pressure. The Mtb-linked HIV antibody enhancement associates with specific mediators important for B cell and antibody development. This Mtb humoral augmentation does not occur due to cross-reactivity, a generalized increase in all antibodies, or differences in duration or amount of antigen exposure. We speculate that the co-localization of Mtb and HIV in lymphatic tissues leads to the emergence of potent HIV antibodies. PWH's Mtb disease status has implications for the future use of HIV broadly neutralizing antibodies as prophylaxis or treatment and the induction of better humoral immunity.

Cross-municipality migration and spread of tuberculosis in South Africa.

Human migration facilitates the spread of infectious disease. However, little is known about the contribution of migration to the spread of tuberculosis in South Africa. We analyzed longitudinal data on all tuberculosis test results recorded by South Africa's National Health Laboratory Service (NHLS), January 2011-July 2017, alongside municipality-level migration flows estimated from the 2016 South African Community Survey. We first assessed migration patterns in people with laboratory-diagnosed tuberculosis and analyzed demographic predictors. We then quantified the impact of cross-municipality migration on tuberculosis incidence in municipality-level regression models. The NHLS database included 921,888 patients with multiple clinic visits with TB tests. Of these, 147,513 (16%) had tests in different municipalities. The median (IQR) distance travelled was 304 (163 to 536) km. Migration was most common at ages 20-39 years and rates were similar for men and women. In municipality-level regression models, each 1% increase in migration-adjusted tuberculosis prevalence was associated with a 0.47% (95% CI: 0.03% to 0.90%) increase in the incidence of drug-susceptible tuberculosis two years later, even after controlling for baseline prevalence. Similar results were found for rifampicin-resistant tuberculosis. Accounting for migration improved our ability to predict future incidence of tuberculosis.

Seroprevalence of Antibodies to SARS-CoV-2 in Rural Households in Eastern Uganda, 2020-2022.

Importance: Estimating the true burden of SARS-CoV-2 infection has been difficult in sub-Saharan Africa owing to asymptomatic infections and inadequate testing capacity. Antibody responses from serologic surveys can provide an estimate of SARS-CoV-2 exposure at the population level. Objective: To estimate SARS-CoV-2 seroprevalence, attack rates, and reinfection in eastern Uganda using serologic surveillance from 2020 to early 2022. Design, Setting, and Participants: This cohort study was conducted in the Tororo and Busia districts of eastern Uganda. Plasma samples from participants in the Program for Resistance, Immunology, Surveillance, and Modeling of Malaria in Uganda Border Cohort were obtained at 4 sampling intervals: October to November 2020, March to April 2021, August to September 2021, and February to March 2022. Each participant contributed up to 4 time points for SARS-CoV-2 serology, with almost half of all participants contributing at all 4 time points, and almost 90% contributing at 3 or 4 time points. Information on SARS-CoV-2 vaccination status was collected from participants, with the earliest reported vaccinations in the cohort occurring in May 2021. Main Outcomes and Measures: The main outcomes of this study were antibody responses to the SARS-CoV-2 spike protein as measured with a bead-based serologic assay. Individual-level outcomes were aggregated to population-level SARS-CoV-2 seroprevalence, attack rates, and boosting rates. Estimates were weighted by the local age distribution according to census data. Results: A total of 1483 samples from 441 participants living in 76 households were tested. Of the 441 participants, 245 (55.6%) were female, and their mean (SD) age was 16.04 (16.04) years. By the end of the Delta wave and before widespread vaccination, adjusted SARS-CoV-2 seroprevalence was 67.7% (95% credible interval [CrI], 62.5%-72.6%) in the study population. During the subsequent Omicron wave, 84.8% (95% CrI, 67.9%-93.7%) of unvaccinated, previously seronegative individuals were infected for the first time, and 50.8% (95% CrI, 40.6%-59.7%) of unvaccinated, already seropositive individuals were likely reinfected, leading to an overall seropositivity of 96.0% (95% CrI, 93.4%-97.9%) in this population. These results suggest a lower probability of reinfection in individuals with higher preexisting antibody levels. There was evidence of household clustering of SARS-CoV-2 seroconversion. No significant associations were found between SARS-CoV-2 seroconversion and gender, household size, or recent Plasmodium falciparum malaria exposure. Conclusions and Relevance: In this cohort study in a rural population in eastern Uganda, there was evidence of very high SARS-CoV-2 infection rates throughout the pandemic inconsistent with national level case data and high reinfection rates during the Omicron wave.

Early Introduction and Rise of the Omicron Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variant in Highly Vaccinated University Populations.

BACKGROUND: The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is highly transmissible in vaccinated and unvaccinated populations. The dynamics that govern its establishment and propensity toward fixation (reaching 100% frequency in the SARS-CoV-2 population) in communities remain unknown. Here, we describe the dynamics of Omicron at 3 institutions of higher education (IHEs) in the greater Boston area. METHODS: We use diagnostic and variant-specifying molecular assays and epidemiological analytical approaches to describe the rapid dominance of Omicron following its introduction into 3 IHEs with asymptomatic surveillance programs. RESULTS: We show that the establishment of Omicron at IHEs precedes that of the state and region and that the time to fixation is shorter at IHEs (9.5-12.5 days) than in the state (14.8 days) or region. We show that the trajectory of Omicron fixation among university employees resembles that of students, with a 2- to 3-day delay. Finally, we compare cycle threshold values in Omicron vs Delta variant cases on college campuses and identify lower viral loads among college affiliates who harbor Omicron infections. CONCLUSIONS: We document the rapid takeover of the Omicron variant at IHEs, reaching near-fixation within the span of 9.5-12.5 days despite lower viral loads, on average, than the previously dominant Delta variant. These findings highlight the transmissibility of Omicron, its propensity to rapidly dominate small populations, and the ability of robust asymptomatic surveillance programs to offer early insights into the dynamics of pathogen arrival and spread.

Viral Dynamics of Omicron and Delta Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Variants With Implications for Timing of Release from Isolation: A Longitudinal Cohort Study.

BACKGROUND: In January 2022, US guidelines shifted to recommend isolation for 5 days from symptom onset, followed by 5 days of mask-wearing. However, viral dynamics and variant and vaccination impact on culture conversion are largely unknown. METHODS: We conducted a longitudinal study on a university campus, collecting daily anterior nasal swabs for at least 10 days for reverse-transcription polymerase chain reaction (RT-PCR) testing and culture, with antigen rapid diagnostic testing (RDT) on a subset. We compared culture positivity beyond day 5, time to culture conversion, and cycle threshold trend when calculated from diagnostic test, from symptom onset, by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant, and by vaccination status. We evaluated sensitivity and specificity of RDT on days 4-6 compared with culture. RESULTS: Among 92 SARS-CoV-2 RT-PCR-positive participants, all completed the initial vaccine series; 17 (18.5%) were infected with Delta and 75 (81.5%) with Omicron. Seventeen percent of participants had positive cultures beyond day 5 from symptom onset, with the latest on day 12. There was no difference in time to culture conversion by variant or vaccination status. For 14 substudy participants, sensitivity and specificity of day 4-6 RDT were 100% and 86%, respectively. CONCLUSIONS: The majority of our Delta- and Omicron-infected cohort culture-converted by day 6, with no further impact of booster vaccination on sterilization or cycle threshold decay. We found that rapid antigen testing may provide reassurance of lack of infectiousness, though guidance to mask for days 6-10 is supported by our finding that 17% of participants remained culture-positive after isolation.

Agreement Between Measures of Adherence to Isoniazid Preventive Therapy Among People With HIV in Uganda.

Background: Isoniazid (INH) preventative therapy is recommended for people with HIV (PWH) in resource-constrained settings. Valid measures are needed to assess adherence. We aimed to examine agreement between measures overall and by level of social desirability. Methods: PWH with latent tuberculosis (TB) were recruited in Mbarara, Uganda. Past 30-day adherence was measured by the number of days with pill bottle openings using a medication event monitoring system (MEMS) and self-reported number of days pills taken. INH concentration (INH plus acetyl INH and their ratio) in hair samples was measured. We used Bland-Altman plots to examine agreement between adherence measures and calculated the area under the receiver operating characteristics curve (AUROC) to determine if INH hair concentration predicted optimal MEMS-measured adherence (≥90%). Results: A total of 301 participants enrolled; 92% were virologically suppressed, and adherence was high. The median (interquartile range [IQR]) number of pill bottle openings in 30 days was 28 (24-30) compared with 30 (28-30) via self-report. The median INH concentration (IQR) was 36.2 (17.2-62.4), and the INH:acetyl ratio was 2.43 (0.99-3.92). Agreement between self-reported and MEMS adherence was greater at more optimal adherence levels. INH:acetyl INH ratio was not predictive of optimal adherence according to MEMS (AUROC, 0.62; 95% CI, 0.52-0.72) in a subset (n = 161). Conclusions: Lower MEMS adherence levels compared with self-report suggest the need for objective adherence measures. Biologic measures have potential, although in this study INH concentration was not predictive of MEMS measured adherence. More data are needed to assess the accuracy of biologic measures.

Methods for handling missing data in serially sampled sputum specimens for mycobacterial culture conversion calculation.

BACKGROUND: The occurrence and timing of mycobacterial culture conversion is used as a proxy for tuberculosis treatment response. When researchers serially sample sputum during tuberculosis studies, contamination or missed visits leads to missing data points. Traditionally, this is managed by ignoring missing data or simple carry-forward techniques. Statistically advanced multiple imputation methods potentially decrease bias and retain sample size and statistical power. METHODS: We analyzed data from 261 participants who provided weekly sputa for the first 12 weeks of tuberculosis treatment. We compared methods for handling missing data points in a longitudinal study with a time-to-event outcome. Our primary outcome was time to culture conversion, defined as two consecutive weeks with no Mycobacterium tuberculosis growth. Methods used to address missing data included: 1) available case analysis, 2) last observation carried forward, and 3) multiple imputation by fully conditional specification. For each method, we calculated the proportion culture converted and used survival analysis to estimate Kaplan-Meier curves, hazard ratios, and restricted mean survival times. We compared methods based on point estimates, confidence intervals, and conclusions to specific research questions. RESULTS: The three missing data methods lead to differences in the number of participants achieving conversion; 78 (32.8%) participants converted with available case analysis, 154 (64.7%) converted with last observation carried forward, and 184 (77.1%) converted with multiple imputation. Multiple imputation resulted in smaller point estimates than simple approaches with narrower confidence intervals. The adjusted hazard ratio for smear negative participants was 3.4 (95% CI 2.3, 5.1) using multiple imputation compared to 5.2 (95% CI 3.1, 8.7) using last observation carried forward and 5.0 (95% CI 2.4, 10.6) using available case analysis. CONCLUSION: We showed that accounting for missing sputum data through multiple imputation, a statistically valid approach under certain conditions, can lead to different conclusions than naïve methods. Careful consideration for how to handle missing data must be taken and be pre-specified prior to analysis. We used data from a TB study to demonstrate these concepts, however, the methods we described are broadly applicable to longitudinal missing data. We provide valuable statistical guidance and code for researchers to appropriately handle missing data in longitudinal studies.

Early immune markers of clinical, virological, and immunological outcomes in patients with COVID-19: a multi-omics study.

Background: The great majority of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) infections are mild and uncomplicated, but some individuals with initially mild COVID-19 progressively develop more severe symptoms. Furthermore, there is substantial heterogeneity in SARS-CoV-2-specific memory immune responses following infection. There remains a critical need to identify host immune biomarkers predictive of clinical and immunological outcomes in SARS-CoV-2-infected patients. Methods: Leveraging longitudinal samples and data from a clinical trial (N=108) in SARS-CoV-2-infected outpatients, we used host proteomics and transcriptomics to characterize the trajectory of the immune response in COVID-19 patients. We characterized the association between early immune markers and subsequent disease progression, control of viral shedding, and SARS-CoV-2-specific T cell and antibody responses measured up to 7 months after enrollment. We further compared associations between early immune markers and subsequent T cell and antibody responses following natural infection with those following mRNA vaccination. We developed machine-learning models to predict patient outcomes and validated the predictive model using data from 54 individuals enrolled in an independent clinical trial. Results: We identify early immune signatures, including plasma RIG-I levels, early IFN signaling, and related cytokines (CXCL10, MCP1, MCP-2, and MCP-3) associated with subsequent disease progression, control of viral shedding, and the SARS-CoV-2-specific T cell and antibody response measured up to 7 months after enrollment. We found that several biomarkers for immunological outcomes are shared between individuals receiving BNT162b2 (Pfizer-BioNTech) vaccine and COVID-19 patients. Finally, we demonstrate that machine-learning models using 2-7 plasma protein markers measured early within the course of infection are able to accurately predict disease progression, T cell memory, and the antibody response post-infection in a second, independent dataset. Conclusions: Early immune signatures following infection can accurately predict clinical and immunological outcomes in outpatients with COVID-19 using validated machine-learning models. Funding: Support for the study was provided from National Institute of Health/National Institute of Allergy and Infectious Diseases (NIH/NIAID) (U01 AI150741-01S1 and T32-AI052073), the Stanford's Innovative Medicines Accelerator, National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA) DP1DA046089, and anonymous donors to Stanford University. Peginterferon lambda provided by Eiger BioPharmaceuticals.